Detection of invasive and native beetle species within trees by chemical analysis of frass

In recent years, several invasive woodborers (Coleoptera: Cerambycidae) have been found in Japan. Aromia bungii is a worldwide important pest of fruits and ornamental species of the genus Prunus. It invaded Japan in the early 2010s and now causes heavy damage to stone fruit trees. Anoplophora glabripennis and Apriona swainsoni are destructive pests of street, ornamental and horticultural trees. The first step in intercepting these beetles is to detect their presence early in their infestation, as accurate identification is crucial for their management. Ejected frass is a major sign of infestation and likely holds information on the insect. We focused on chemicals in both larvae and frass, and conducted a GC–MS analysis of these three invasive beetles and the native Anoplophora malasiaca. In all four species, 4 or 5 species-specific hydrocarbons were detected in both larvae and frass. These results indicate that analysis of hydrocarbons in frass could allow definitive detection of invasive wood-boring pests.


Scientific Reports
| (2023) 13:11837 | https://doi.org/10.1038/s41598-023-38835-x www.nature.com/scientificreports/ other hand, frass ejection from trees infested by pest insects is often observed in the field. Frass includes woody materials and sometimes faeces of infesting insects. Invasive beetles can be identified from their genetic information. Analysis of frass by real-time PCR confirmed the presence of A. bungii 25 and another wood-boring pest 26 . Although this method guarantees accuracy of species identification, the stability of genetic information in the field is not assured under the tough conditions of rain and sunlight. To develop better methods for identifying infesting beetles under tough field conditions, we focused on the hydrocarbons in their frass. Among possible chemical components in frass, hydrocarbons are synthesized by insects and are chemically more stable than polar compounds and DNA. Insect cuticular hydrocarbons (CHCs) have roles in maintaining the insect's water balance and acting as signalling molecules for mate recognition and chemical communication [27][28][29] . CHCs of many adult insects are reported to be species specific 30 . In contrast, only a few reports of larval CHCs were found: one on blowfly larvae 31 , one on Tribolium confusum (Coleoptera, Tenebrionidae) 32 , and some on parasitic lepidopteran larvae which penetrate ant nests 33 . These reports describe age-dependent changes in larval CHCs (blowfly), kairomonal activity (T. confusum), or chemical camouflage.
Here, we compared profiles of CHCs of larvae and frass of three invasive and one native beetles and confirmed these commonalities. Based on these comparisons, we could reveal that analysis of hydrocarbons in frass could contribute to detecting beetle species in trees. Furthermore, we demonstrate the validity of our method by using frass samples of A. bungii. We discuss the possible use of hydrocarbon analysis for the identification of infesting species.

Results
Common hydrocarbons detected in larvae and frass of four beetle species. Table 1 lists hydrocarbons commonly detected in frass and larvae of each species, with the Kovát's Index and molecular or fragment ions used for their identification. Double-bond positions of unsaturated hydrocarbons were determined only in A. bungii. Some hydrocarbons were detected in multiple species; for example, n-tricosane was detected in A. bungii, A. glabripennis and A. swainsoni. All four species have species specific hydrocarbons: for example, 6,9-C 25:2 and 6,9-C 27:2 in A. bungii; C 27:1 in A. glabripennis; C 28 , 4Me-C 28 and C 29 in A. malasiaca; and C 23:1 and C 25:1 in A. swainsoni.

Hydrocarbon analysis of sawdust and frass from plum tree infested by A. bungii.
Larval-specific hydrocarbons were not detected in the sawdust extract (Fig. 6a). Only frass showed the five characteristic hydrocarbons (Fig. 6b, arrows).

Time-course hydrocarbon analysis of Aromia bungii frass. Weekly analysis of A. bungii frass showed
that the hydrocarbon profiles were unchanged over 5 weeks after larvae hatching (Fig. 7a, week 1; b, week 3; c, week 5). As a reference data, field-collected frass ejected by over-wintered, 9-month larvae included same species-specific hydrocarbons (Fig. 7d). Aromia bungii-specific hydrocarbon profiles were detected through the larval period.

Discussion
We developed a new method to detect three invasive and one native beetle species from frass ejected from infested trees. Extraction of hydrocarbons and GC-MS analysis correctly identified the beetle species in the trees. Every sample showed clear total ion chromatograms, and all detected peaks could be chemically characterized. Four or five hydrocarbons common to both larvae and frass were consistently detected within species. As reported of other insect species 28 , CHC profiles of tested larvae differed among species. We also got the same hydrocarbon profiles of A. bungii frass from laboratory ( Fig. 1) and field samples (Fig. 5). This result reveals that our method could be applied to both laboratory and field samples. CHCs of insects consist of a complex mixture of straight-chain (saturated), unsaturated, and methyl-branched components with over 20 carbon atoms 28 . In A. bungii, A. malasiaca and A. glabripennis, chemical profiles of CHCs of adults and their larvae are not common [ 34 (cf. our data), 35,36 ]. There is no information on the roles of larval CHCs of these beetles in chemical communication, but some may have kairomonal activity against parasitoids. Dastarcus helophoroides (Coleoptera: Bothrideridae) is a larval predator of cerambycid forest tree pests, and its mass release is one option for controlling beetles 37 . Volatile chemicals from the host tree or insects play a key role in host searching from a distance 37 , although, commonly detected CHCs in this study are slightly volatile and almost non-volatile, there is a possibility to have close-distant or contact kairomonal activity.
Currently, the most accurate method to identify infesting beetle species is to collect larvae and rear them to adulthood, because beetle larvae look very similar 38 . However, this method damages trees. Instead, if ejected frass is present, hydrocarbon analysis could be used to identify them without further damage to the tree. We confirmed the validity of our method with A. bungii because of sample availability. The results of the chemical analysis of A. bungii frass from plum and peach (Figs. 1b, 5b), plum (Fig. 6b), and cherry trees (Fig. 5a) show that our method can work with different tree species. Even within their 'first week of feeding or just after hatch, frass was seen. Only 1 mg equivalent of frass extract injected into the GC/MS could determine the profiles of its typical hydrocarbons. Thus, our method could be applied very early in larval infestation; the earlier an infestation can be detected, the earlier it can be treated, avoiding further damage. This is crucial to reducing the damage by wood-boring beetles to economically, culturally or historically important trees. Throughout our experiments, larvae-frass common hydrocarbons were stably detected from early stage (1-5 weeks, Fig. 7a-c), 2-month over (Fig. 5a,b) and 9-month over (Fig. 7d). As is known in other insects, CHCs can change over the larval stage 31 . If the same tendency existed in the beetles, our method could allow us to estimate larval age or size. The entire larval stage changes of their CHCs composition should be investigated in our future work. In the case of A. glabripennis and A. malasiaca, however, the frass sometimes did not include detectable amounts of hydrocarbons. In our interpretation, the difference may be caused by larval behaviour. Aromia bungii and species of Apriona (including A. swainsoni) continually make excretory holes for ejecting their frass 39,40 . During our preliminary experiment, Aromia larvae used their body segments to eject the frass. This active ejection of frass may transfer their CHCs to the frass. On the other hand, this behaviour has not been reported in Anoplophora larvae and we haven't observed it. We could detect A. malasiaca-specific hydrocarbons in frass from willow twigs (Fig. 3), but not in other trials using fresh or live trees. One possibility is that when feeding on spatially restricted dry material (such as cut twigs), A. malasiaca larvae have to maintain their living and feeding space by ejecting frass. As a result of that behaviour, their CHCs might often be undetectable.
Many other sympatric insects eject frass in the same way as our beetles. To improve risk management of trees worldwide, we should accumulate examples of insect cuticular and frass hydrocarbon profiles not only of Coleopteran but also of other frass-ejecting insect species.  Frass collection. Aromia bungii. In the laboratory, small branches of plum or peach (5 cm diam., 10 cm long) were partially wrapped in 1-cm-wide Parafilm (Bemis Flexible Packaging, Chicago, IL, USA), and females of A. bungii were housed on them for 2 days. Eggs laid between the bark and the Parafilm were collected and laid on wet filter paper (9 cm diam., Toyo Roshi Kaisha, Ltd, Tokyo, Japan). Eggs on filter paper were placed together in a plastic Petri dish (9 cm diam. × 2 cm height) at 24 °C under a 15L:9D photoperiod, illuminated by fluores-  www.nature.com/scientificreports/ cent lamps for 10 days. Newly hatched larvae were placed on new twigs of plum. After a few weeks, larvae began to eject frass, which was then collected by forceps into glass vials weekly for 5 weeks. Frass was also collected from peach groves and cherry trees in Sano and Ashikaga city on October 4th, 2022, Tochigi prefecture, where we collected the A. bungii. These field collected frass were expected to be ejected by over 2-month larvae of A. bungii. Frass of over-wintered larvae also collected on May 25th, 2022 from woods bring back from peach groves in Sano city in December 2021 and maintained in the laboratory (9-month over larvae).

Materials and methods
Anoplophora glabripennis and A. malasiaca. Females of each species were individually kept in clear plastic cups (ca. 11 cm diam. × 9.5 cm height) together with host twigs, mainly willow (3 cm diam., 5 cm long). Eggs laid under the bark were collected and laid on wet filter paper. Eggs on filter paper were placed together in a plastic Petri dish as above for 10 days. Newly hatched larvae were placed on new plum or willow twigs. Ejected frass was collected as above in our laboratory (A. glabripennis and A. malasiaca, three samples) and from two frass samples were collected from Cercidiphyllum japonicum in Tsukuba from the field (A. glabripennis).
Apriona swainsoni. Males and females of A. swainsoni were kept together in small mesh cages (28 cm × 30 cm × 18 cm). Females laid eggs into Maackia Amurensis branches. The branches were maintained as above. After a few weeks, larvae began to eject frass, which was collected as above in our laboratory (two samples) in 2022. In 2023, three larvae and frass were collected from the infested wood cut in Fukushima prefecture.
Extraction and purification of hydrocarbons from frass and larvae. All frass was freeze-dried for 16 h at − 30 °C before extraction. Dried samples were weighed and then extracted in 5-8 mL of n-hexane (HPLC grade, Fujifilm-Wako, Osaka, Japan) for 5 min. Sample sizes of each frass were 10 for A. bungii, 5 for A. glabripennis and A. swainsoni, 3 for A. malasiaca. Larvae were placed on filter paper for 20 min to void faeces and then frozen (− 30 °C). Each larva was put into a glass vial (5-12 mL) and extracted with n-hexane for 5 min. Sample sizes of each larva were 5 for A. bungii, 5 for A. glabripennis, 3 for A. malasiaca, and 5 for A. swainsoni. www.nature.com/scientificreports/ Hexane extracts were filtered and poured through a silica gel column (Wakogel C-300, 0.5 g, Fujifilm-Wako). The final volume of extracts after concentration depended on frass volume or larval size, but 0.001 g equivalent of frass sample or 0.01 larva equivalent dissolved in 1 µL n-hexane was injected into the GC-MS.

Comparison of hydrocarbons in frass and larvae on plum tree in the laboratory. Females of A.
bungii were introduced into a meshed cage with a plum tree in a pot (2 years old, 100 cm high) and allowed to lay freely for 2 days. After 2-3 weeks, hatched larvae started to eject frass. At the same time, sawdust was collected by hand-sawing for analysis to confirm the components of the host plant. Frass and sawdust were extracted as above.

Gas chromatography/mass spectrometry (GC/MS) analyses. GC/MS analyses were performed on
an Agilent 7890N GC system (Agilent, Santa Clara, CA, USA) interfaced to a JEOL JMS-T100GC time-of-flight mass spectrometer (JEOL, Tokyo, Japan) in EI mode at 70 eV at 220 °C. Samples were injected in splitless mode at 220 °C for 1 min, with helium as the carrier gas in constant flow mode (1.1 mL/min). The capillary column, a DB-5MS (30 m × 0.25 mm ID × 0.25 μm film thickness; Agilent), was linked to an MS, and the GC oven temperature was held at 40 °C for 1 min, increased from 40 to 250 °C at 10 °C/min and then held at the final temperature for 10 min.

Identification of hydrocarbons in larvae and frass.
Hydrocarbons were identified by the mass spectrum and the Kovát index of each peak 41 . A reference hydrocarbon mixture that included saturated, evennumbered hydrocarbons from C 12 to C 32 was analysed as above, with reference to KIs of standard compounds. Only A. bungii yielded diene components in sufficient amount for derivatization to decide the position of two double bonds. Diene components were partially reduced with hydrazine and epoxidated by m-chloroperoxybenzoic acid. The resultant epoxide mixture was analysed by GC-MS, and the position of the double bonds was determined 42 .

Data availability
The data presented in this study are available on request from the corresponding author.